Abstract Zika virus is a burgeoning global threat. This R21 project responds to NOT-AI-16-026 Interest to Highlight High-Priority Zika Virus (ZIKV) Research Areas. Adenoviral (Ad) vectors are arguably one of the most potent vaccine vectors available today. For example, we demonstrated in a head to head comparison against licensed influenza protein vaccine, replication-defective Ad (RD-Ad) vaccines mediated superior protection against heterologous influenza challenge. In direct comparison in non-human primates, RD-Ad vaccines were more robust than DNA or vaccinia vaccines. While RD-Ad vaccines can be potent when compared to other vaccines, RD-Ad vaccines are actually not the most potent type of Ad vaccine. Most are RD-Ads that are deleted for their E1 gene to block replication primarily to avoid the Ad from causing an actual adenovirus infection in the recipient. An E1-deleted Ad vaccine infects a cell, delivers its one copy of an antigen gene, and express 1X of this protein. In contrast, replication- competent Ads (RC-Ads) retain their E1 gene and can replicate their DNA and antigen genes they carry 10,000-fold in an infected cell. RC-Ads can produce 100's of times the amount of protein and drive substantially stronger immune responses than E1-deleted vectors While RC-Ad are potent, fully replication- competent vaccines can cause frank adenovirus infections in vaccinees or healthcare workers. New Single-cycle Ad (SC-Ad) Vaccines. To harness antigen gene replication and amplified immune responses, but avoid the risk of Ad infection, we recently developed SC-Ads. SC-Ads are engineered to allow them to still replicate their DNA and antigen genes like an RC-Ad, but they cannot produce infectious Ads. In head to head comparisons, SC-Ad required 33 times less vector than RD-Ad to drive the same level of protein expression. When RD- and SC-Ads expressing influenza hemagglutinin (HA) were used for single intranasal immunization, 100-fold lower doses of SC-Ad were needed to generate hemagglutination inhibition (HAI) antibodies than RD-Ad. This suggests that a single production of SC-Ad may be able to vaccinate 100X as many people than RD-Ads. After single intranasal immunization, SC-Ads generate antibodies against HA or against Ebola glycoprotein that rise over months and remain elevated for more than 6 months (and counting). We therefore propose that SC-Ad vaccines are a promising vector platform to combat the emerging Zika virus epidemic. Further, we propose deploying this vaccine as a mucosal vaccine will provide a simple needle-free platform for global vaccination in the face of this potential pandemic. We will test SC-Ad utility against Zika virus in the following aims: Aim 1. Generate and Test SC-Ad Vaccines to Drive Immune Responses against Zika Virus. Aim 2. Test the Ability of SC-Ad Vaccines to Protect Against Zika Virus Challenge in Squirrel Monkeys. Successful testing of the SC-Ad platform against Zika virus will provide key preclinical data to begin translating this new robust vaccine platform for deployment as a simple global vaccine.